Project Summary/Abstract Combinatorial biosynthesis and metabolism studies of novel tetracenomycins Anthracyclines are natural product antibiotics that are among the most effective anticancer drugs used in the clinic. Biosynthetic modification of anthracyclines is a promising strategy to generate new chemical analogs with differentiated anticancer activities and expanded therapeutic windows. Elloramycin (ELM, 1) is an anthracycline antibiotic produced by Streptomyces olivaceus T 2353 that features a tetracyclic elloramycinone aglycone and an appended 8-O-2,3,4-tri-O-methyl-a-L-rhamnose sugar. Elloramycins and tetracenomycins (TCMs) exhibit mild antiproliferative activity due to inhibition of DNA topoisomerase II. Furthermore, optimization of the deoxysugar moiety is essential for refining the cellular penetration, potency, clearance, and metabolism of the analogs. However, we still do not fully understand the structure-activity-relationships of the TCM deoxysugar on antiproliferative activity and drug metabolism. Furthermore, despite preparation of >20 different analogs of ELM, the SAR of these analogs has not yet been assessed rigorously in a panel of cancer cell lines. Our long-term goal is to produce anthracycline analogs with improved antiproliferative activity. To facilitate production of analogs, we will exploit the S. lividans (cos16F4) expression system to alter the deoxysugar moiety of tetracenomycins via combinatorial biosynthesis. This comprehensive platform will facilitate development of new drug leads for use in human cancer. The overall objectives of the proposed research are three-fold: (1) to synthesize novel TCM derivatives, (2) to validate the anticancer activity of the TCMs, and (3) to develop a S9 fraction assay to evaluate metabolism of 1 and the most active analogs. Our hypotheses are that (1) ElmGT will be ?substrate-flexible? enough to transfer the intended TDP-deoxysugar donors to 8-demethyl-tetracenomycin C, (2) the appended deoxysugar will alter the anticancer activity via binding to topoisomerase II and DNA, and (3) it will alter drug metabolism via differential binding to hepatic cytochrome P450s. Our specific aims will test these hypotheses: (Aim 1) we will heterologously express ?sugar plasmids? in S. lividans (cos16F4) to alter the TCM deoxysugar moiety, (Aim 2) we will evaluate the antiproliferative activity of the new TCM analogs, (Aim 3) we will develop a S9 fraction assay to determine the metabolic profile of TCM analogs. The rationale is that the ?substrate-flexible? glycosyltransferase, ElmGT, should accept the novel TDP-deoxysugar donors. This contribution is significant because the expected production platform will more fully explore the chemical space of TCM analogs. Furthermore, this research is innovative because we are investigating the impact of deoxysugar modifications on antitumoral activity and metabolism of tetracenomycins, drug properties which are currently not well-understood.